The present invention relates generally to systems and methods for processing data representing sigmoid type curves or growth curves, and more particularly to systems and methods for detecting and correcting data spikes in real-time Polymerase Chain Reaction (PCR) amplification curves and other sigmoid or growth-type curves.
The Polymerase Chain Reaction (PCR) is an in vitro method for enzymatically synthesizing or amplifying defined nucleic acid sequences. The reaction typically uses two oligonucleotide primers that hybridize to opposite strands and flank a template or target DNA sequence that is to be amplified. Elongation of the primers is catalyzed by a heat-stable DNA polymerase. A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in an exponential accumulation of a specific DNA fragment. Fluorescent probes or markers are typically used in real-time PCR, or kinetic PCR, to facilitate detection and quantification of the amplification process.
A typical kinetic PCR curve is shown in FIG. 2, where fluorescence intensity values (y-axis) are plotted vs. cycle number (x-axis) for a typical PCR process. In this case, the formation of PCR products is monitored in each cycle of the PCR process. The amplification is usually measured in thermocyclers which include components and devices for measuring fluorescence signals during the amplification reaction. An example of such a thermocycler is the Roche Diagnostics LightCycler (Cat. No. 20110468). The amplification products are, for example, detected by means of quenched fluorescently labeled hybridization probes which only emit fluorescence signals after they are bound to a target nucleic acid sequence and subsequently degraded by the 5′ to 3′ nuclease activity of a DNA polymerase. Other examples include fluorescent signals generated during nucleic acid amplification where fluorescent dyes bind to double-stranded DNA and experience an increase in their fluorescence quantum yield.
During PCR data acquisition, artifacts such as air bubbles, gamma rays or incomplete mixing can cause outlier spikes in growth curves. An outlier spike or “spike” is a sudden jump in the signal that doesn't reflect the real growth. Spikes can affect further calculations done on the growth curves and therefore should be detected and corrected. FIG. 2 shows an example of a PCR growth curve including outlier spikes.
Current spike removal systems are available. However, these spike removal systems generally suffer from major deficiencies, such as an inability to correct most of the spikes wider than one cycle, an inability to correct early and late spikes, and a poor ability to detect and correct spikes in other various situations. Current spike removal systems also tend to have parameters that are often difficult to set.
Therefore it is desirable to provide outlier spike removal systems and methods that overcome the above and other problems. The systems and methods should provide a spike removal process that is more reliable and that includes fewer parameters or is parameter free.